Therapeutic strategies for acute lung injury (ALI), a major cause of morbidity and mortality in critically ill patients, are extremely limited. A defining feature of ALI and a target for intervention is the disruption of the lung vascular endothelial cell (EC) barrier which results in leakage of fluid, protein and cells into the airspaces of the lungs. Our novel studies indicate that the extracellular matrix component, hyaluronan (HA), and its cell surface receptor family, CD44, are important in normal EC function and angiogenesis. We have demonstrated that the ligation of the CD44 isoform, CD44s (standard form) by high molecular weight HA (HMW-HA) results in Rac1-dependent cortical actin formation and EC barrier enhancement. In contrast, low molecular weight HA ((LMW-HA), produced in pathological states by hyaluronidase enzymes) occupies CD44 variant 10 (CD44v10) and mediates RhoA-dependent actin stress fiber formation and EC barrier disruption. Therefore, enhancing the effects of HMW-HA/CD44s signaling pathways and diminishing the effects of LMW-HA/CD44v10 signaling may provide a novel therapeutic strategy for ALI via enhancing pulmonary vascular integrity. Specific Aim #1 will expand our published and preliminary data by determining HMW-HA/CD44s regulation of EC barrier function by specialized structures called caveolin-enriched microdomains (CEM) containing caveolin-1 and dynamin 2. In Specific Aim #2, we will determine HMW-HA-mediated protection from pulmonary EC barrier disruption through inhibition of RhoA GTPase and activation of Rac1 GTPase. Finally, Specific Aim #3 will employ therapeutic strategies designed to further validate HA/CD44 regulation of vascular integrity (CD44v10-specific blocking antibodies, hyaluronidase and Rho Kinase inhibitors, siRNA) using ACE antibody-directed nanocarrier technologies to target the pulmonary endothelium. These studies will yield novel mechanistic insights into HMW- HA's protection from ALI-associated pathobiology and inflammatory lung injury as well as provide novel therapeutic strategies to reduce ALI-mediated lung injury.